Mutations in the RAS family of small GTPases are responsible for approximately 30% of human tumors. Despite intense activity over the last 25 years, the identification of direct inhibitors of RAS has proven challenging, necessitating alternative approaches to modulating RAS activity. One such approach takes advantage of the apparent dependency of mutant RAS-dependent cells on non-oncogenic cellular factors. For instance, the serine/threonine kinase, STK33, has recently been shown by RNAi techniques to be required for the survival and proliferation of mutant KRAS-dependent cancer cells, but not KRAS-independent cells. These results indicate a co-dependency between mutant KRAS and STK33 that results in a synthetic lethal interaction upon suppression of STK33. The overall goal of this proposal is to identify a set of direct STK33 kinase activity inhibitors that can recapitulate the exquisite cell-based specificity of RNAi for inducing apoptosis in mutant KRAS-dependent cells without discernable effects on KRAS-independent cells. The specific aims of this proposal are to (1) identify STK33 kinase activity inhibitors in a robust, sensitive, previously validated primary screen, (2) utilize a panel of secondary cell-based assays to select for those STK33 inhibitors that specifically target mutant KRAS-dependent cells, and (3) implement a critical path to facilitate probe optimization and development. The longer term objective of this research program is to utilize these STK33- specific probes in conjunction with a comprehensive proteomics approach to elucidate the molecular basis of RAS-dependent regulation and mutant KRAS-dependent tumorigenesis with the goal of translating this knowledge into a therapeutic opportunity. PUBLIC HEALTH RELEVANCE: The overall goal of this research is to identify chemical probes that will inform us about how the most common mutation occurring in human tumors causes cancer. This knowledge along with these chemical probes as lead structures can then be used to generate novel therapeutic opportunities.